Cell integrity markers for in vitro evaluation of cytotoxic responses to bacteria-containing commercial insecticides

Toxicity of two commercial "BT" products, containing Bacillus thuringiensis subsp. kurstaki spores (Btk) and associated parasporal inclusion body proteins, was tested in vitro using two unrelated lepidoperan cell lines and several markers of cell integrity (morphology, quantification of loss of adherence and electron transport (redox) activity, and degradation of nuclear DNA, actin, hsp-70, and beta-tubulin). With doses of 10(-7), 10(-5), and 10(-3) International Units (IU)/target cell, these markers measured exposure-dependent effects closely linked to cell death, which occurred rapidly once Btk spores germinated, unless inhibited by antibiotic. Derivation of marker half-lives (HL50) revealed that temperature critically affected product performance. Between 34 and 37 degrees C, HL50 was < or = 5 hr, but dose discrimination between 10(-5) and 10(-3) IU was poor. At temperatures less than 34 degrees C, the resolution between different HL50s and doses increased in a manner directly relating to published data obtained from in vivo BT-spore-induced LD50 assays. It was concluded that BT product toxification is complex, essentially enabled by an autobiotransformation process in which dose-response lag is affected by temperature-dependent temporal expression of spore germination and critical buildup of vegetative cells and byproduct toxicants. The in vitro dosimetry assays described here are potentially useful for obtaining mechanistic toxicologic data and in vivo relevant quantifications of subingredient activities in various commercial BT formulations as well as in other microbe-based biotechnology products.